Fluorescence Resonance Energy Transfer Between Donor-Acceptor Pair on Two Oligonucleotides Hybridized Adjacently to a DNA Template
Lili Wang, Adolfas K. Gaigalas, J L. Blasic, Marcia J. Holden, David T. Gallagher, R Pires
We have used fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides, with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For FRET, both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration has been implemented for quantitative real-time PCR with the LightCycler technology, where a 1 to 5 base separation between donor and acceptor was recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 ). The results show that when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80% although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling has been used to predict the structure of these three-component constructs. The duplex mostly retains normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Since efficient and reproducible energy transfer is observed over the distance range investigated here, we demonstrate that FRET can be quantified in a broad range of distances in real-time PCR.
fluorescein, fluorescence resonance energy transfer, hybridization probes, quantitative PCR, rhodamine, three-component DNA construct
, Gaigalas, A.
, Blasic, J.
, Holden, M.
, Gallagher, D.
and Pires, R.
Fluorescence Resonance Energy Transfer Between Donor-Acceptor Pair on Two Oligonucleotides Hybridized Adjacently to a DNA Template, Biopolymers
(Accessed December 1, 2023)